Conidial melanin of the human pathogenic fungusAspergillus fumigatusdisrupts cell autonomous defenses in amoebae

Abstract

19The human pathogenic fungus Aspergillus fumigatus is a ubiquitous saprophyte that 20 causes fatal infections in immunocompromised individuals. Following inhalation, conidia 21 are ingested by innate immune cells and can arrest phagolysosome maturation. How 22 such general virulence traits could have been selected for in natural environments is 23 unknown. Here, we used the model amoeba Dictyostelium discoideum to follow the 24 antagonistic interaction of A. fumigatus conidia with environmental phagocytes in real 25 time. We found that conidia covered with the green pigment 1,8-dihydroxynaphthalene-26 (DHN)-melanin were internalized at far lower rates when compared to those lacking the 27 pigment, despite high rates of initial attachment. Immediately after uptake of the fungal 28 conidia, nascent phagosomes were formed through sequential membrane fusion and 29 fission events. Using single-cell assays supported by a computational model integrating 30 the differential dynamics of internalization and phagolysosome maturation, we could 31 show that acidification of phagolysosomes was transient and was followed by 32 neutralization and, finally, exocytosis of the conidium. For unpigmented conidia, the 33 cycle was completed in less than 1 h, while the process was delayed for conidia covered 34 with DHN-melanin. At later stages of infection, damage to infected phagocytes triggered 35 the ESCRT membrane repair machinery, whose recruitment was also attenuated by 36 DHN-melanin, favoring prolonged persistence and the establishment of an intracellular 37 germination niche in this environmental phagocyte. Increased exposure of DHN-melanin 38 on the conidial surface also improved fungal survival when confronted with the 39 fungivorous predator Protostelium aurantium, demonstrating its universal anti-40 phagocytic properties.41 42 3 Keywords 43 Aspergillus fumigatus, Dictyostelium discoideum, Protostelium aurantium, DHN-melanin, 44 phagocytosis, phagosome maturation, acidification, v-ATPase, ROS, NADPH oxidase, 45 membrane damage, membrane repair, ESCRT machinery. 46 et al., 2013, Hillmann et al., 2015, Mattern et al., 2015. Waeyenberghe 87D. discoideum is a professional soil phagocyte that constantly engulfs microbes for food 88 and thus has to protect itself from potentially harmful intracellular pathogens (Cosson 89 and Soldati, 2008, Dunn et al., 2018). To avoid infection, the phagocytic host has to 90 eliminate pathogens by forming a functional phagosome before they can escape or 91 establish a survival niche. After engulfment, the pathogen is trapped inside the nascent 92 phagosome, which is mainly derived from the plasma membrane. Initially, it lacks any 93 microbicidal capacity that is essential for pathogen control. By a sequence of membrane 94 6 fusion and fission events, the phagosome acquires its full range of antimicrobial and 95 degradative features. This conversion is known as phagosome maturation, during which 96 the compartment undergoes consecutive fusion with early and late endosomes and 97 lysosome...